Chondromodulin is necessary for cartilage callus distraction in mice.

Chondromodulin (Cnmd) is a glycoprotein known to stimulate chondrocyte growth. We examined in this study the expression and functional role of Cnmd during distraction osteogenesis that is modulated by mechanical forces. The right tibiae of the mice were separated by osteotomy and subjected to slow progressive distraction using an external fixator. In situ hybridization and immunohistochemical analyses of the lengthened segment revealed that Cnmd mRNA and its protein in wild-type mice were localized in the cartilage callus, which was initially generated in the lag phase and was lengthened gradually during the distraction phase. In Cnmd null (Cnmd-/-) mice, less cartilage callus was observed, and the distraction gap was filled by fibrous tissues. Additionally, radiological and histological investigations demonstrated delayed bone consolidation and remodeling of the lengthened segment in Cnmd-/- mice. Eventually, Cnmd deficiency caused a one-week delay in the peak expression of VEGF, MMP2, and MMP9 genes and the subsequent angiogenesis and osteoclastogenesis. We conclude that Cnmd is necessary for cartilage callus distraction.

TRPV4-pathy manifesting both skeletal dysplasia and peripheral neuropathy: a report of three patients.

Heterozygous missense mutations of transient receptor potential vanilloid 4 channel (TRPV4) cause a spectrum of skeletal disorders, including brachyolmia, spondylometaphyseal dysplasia Kozlowski type, metatropic dysplasia, parastremmatic dysplasia, and spondyloepimetaphyseal dysplasia Maroteaux type. Similarly, heterozygous missense mutations of TRPV4 cause a spectrum of peripheral neuropathy, including hereditary motor and sensory neuropathy type IIC, congenital spinal muscular atrophy, and scapuloperoneal spinal muscular atrophy. There are no apparent differences in the amino acid positions affected or type of change predicted by the TRPV4 mutations responsible for the two disease spectrums; nevertheless, no fundamental phenotypic overlap has been shown between the two spectrums. Here, we report on three patients who had both skeletal dysplasia and peripheral neuropathy caused by heterozygous TRPV4 missense mutations. The skeletal and neurologic phenotypes of these patients covered the wide spectrum of reported TRPV4-pathies (disease caused by TRPV4 mutations). The molecular data are complementary, proving that "neuropathic" mutations can cause skeletal dysplasia but also the "skeletopathic" mutations can lead to neuropathies. Our findings suggest that pathogenic mechanisms of TRPV4-pathies in skeletal and nervous systems are not always mutually exclusive and provide further evidence that there is no clear genotype-phenotype correlation for either spectrum. Co-occurrence of skeletal dysplasia and degenerative neuropathy should be kept in mind in clinical practice including diagnostic testing, surgical evaluation, and genetic counseling.

CANT1 mutation is also responsible for Desbuquois dysplasia, type 2 and Kim variant.

BACKGROUND: Desbuquois dysplasia (DD) is a recessively inherited condition characterised by short stature, generalised skeletal dysplasia and advanced bone maturation. DD is both clinically and radiographically heterogeneous, and two subtypes have been distinguished based on the presence (type 1) or absence (type 2) of an accessory metacarpal bone. In addition, an apparently distinct variant without additional metacarpal bone but with short metacarpals and long phalanges (Kim variant) has been described recently. Mutations in the gene that encodes for CANT1 (calcium-activated nucleotidase 1) have been identified in a subset of patients with DD type 1. METHODS: A series of 11 subjects with DD from eight families (one type 1, two type 2, five Kim variant) were examined for CANT1 mutations by direct sequencing of all coding exons and their flanking introns. RESULTS: Eight distinct mutations were identified in seven families (one type 1, one type 2 and all 5 Kim variant): three were nonsense and five were missense. All missense mutations occurred at highly conserved amino acids in the nucleotidase conserved regions of CANT1. Measurement of nucleotidase activity in vitro showed that the missense mutations were all associated with loss-of-function. CONCLUSION: The clinical-radiographic spectrum produced by CANT1 mutations must be extended to include DD type 2 and Kim variant. While presence or absence of an additional metacarpal ossification centre has been used to distinguish subtypes of DD, this sign is not a distinctive criterion to predict the molecular basis in DD.

A founder mutation of CANT1 common in Korean and Japanese Desbuquois dysplasia.

Desbuquois dysplasia (DBQD) is a severe skeletal dysplasia of autosomal recessive inheritance. DBQD is classified into types 1 and 2 based on presence or absence of hand anomalies. In a previous study, we found a CANT1 (for calcium-activated nucleotidase 1) mutation, c.676G>A in five DBQD families. They were all East Asians (Japanese or Korean). The high prevalence of the same mutation among Japanese and Korean suggested that it is a common founder mutation in the two populations. To examine a possible common founder, we examined the region around CANT1 in chromosomes with c.676G>A mutation by genotyping polymorphic markers in the region for the families. We examined their haplotypes using the family data. We identified in all families a common haplotype containing the CANT1 mutation that ranged up to 550 kb. The two unrelated carriers of the mutation in general populations in Korea and Japan could also have the haplotype. We estimated the age of the founder mutation as ∼ 1420 years (95% CI=880-1940 years). The c.676G>A mutation of CANT1 commonly seen in Japanese and Korean DBQD should be derived from a common founder.

A variant of Desbuquois dysplasia characterized by advanced carpal bone age, short metacarpals, and elongated phalanges: report of seven cases.

We present the clinical and radiological findings of seven patients with a seemingly new variant of Desbuquois dysplasia (DBQD) and emphasize the radiographic findings in the hand. All cases showed remarkably accelerated carpal bone ages in childhood, but none of the patients had an accessory ossification center distal to the second metacarpal, or thumb anomalies, instead, there was shortness of one or all metacarpals, with elongated appearance of phalanges, resulting in nearly equal length of the second to fifth fingers. The two sibs followed for 20 years showed narrowing and fusion of the intercarpal joints with age and ultimately, precocious degenerative arthritis. The changes in the feet were similar to those of the hands, with advanced tarsal bone ages, shortness of the metatarsals and elongation of the second and third toes. Other radiographic findings were narrowness of the intervertebral disc spaces resulting in precocious degenerative spondylosis and progressive scoliosis. The femoral neck was short and thick and showed a persistent enlargement of the lesser trochanter with a high-riding, bulbous greater trochanter that became more prominent with age. Molecular testing of the diastrophic dysplasia sulfate transporter (DTDST) gene was performed on six patients and no mutations were detected. This radiographic and clinical observation further adds to the evidence that there may be subtypes of DBQD. Long-term follow-up showed that severe precocious osteoarthritis of the hand and spine is a major manifestation of this specific variant.

[Genetic basis for skeletal disease. Genetic defects in chondrodysplasia].

Chondrodysplasia is a subset of skeletal dysplasia caused by genetic defects affecting chondrogenesis and its development, showing abnormal shape and structure of the skeleton. Pathology of growth plate results in defective skeletal development, such as short stature, while pathology of articular cartilage predisposes degenerative skeletal disease, such as early-onset osteoarthritis. Recently identified genetic basis for chondrodysplasia contributed much in understanding the biology and pathology of cartilage. The accumulated knowledge would be a clue to develop fundamental treatment for chondrodysplasia.

Czech dysplasia occurring in a Japanese family.

Czech dysplasia (OMIM 609162) is a recently established COL2A1 disorder characterized by normal height, early-onset osteoarthritis, platyspondyly, short metatarsals, and the absence of ophthalmological complications or cleft palate. A specific missense mutation (c.823C > T, R275C) in the exon 13 of the COL2A1 gene, coding for the triple helical domain of the alpha 1 chain of the type II collagen, has been linked to Czech dysplasia, which is quite a unique situation among the COL2A1 disorders. Since all of the 11 families and patients reported to date were of European ancestry, an ancient single origin of the R275C mutation was speculated about. Here we report on a Japanese family consisting of three patients with Czech dysplasia, each member showing valgus knees in addition to remarkably uniform manifestation of the clinical and radiological abnormalities. Mutation analysis documented the COL2A1 c.823C > T mutation in all affected individuals. In conclusion, this report provides novel evidence for the independent occurrence of Czech dysplasia among the populations.

Expression of HMGA2-LPP and LPP-HMGA2 fusion genes in lipoma: identification of a novel type of LPP-HMGA2 transcript in four cases.

BACKGROUND: In a subset of lipoma, a specific t(3;12)(q27-28;q14-15) chromosomal translocation leads to the fusion of the high mobility group A2 (HMGA2) gene and the lipoma preferred partner (LPP) gene. Although the expression of HMGA2-LPP fusion gene has been reported in lipomas, the reciprocal LPP-HMGA2 fusion gene has rarely been described. MATERIALS AND METHODS: Ninety-eight cases of lipoma were analyzed for the possible expression of HMGA2-LPP and LPP-HMGA2 fusion genes using a reverse-transcription polymerase chain reaction method. RESULTS: Ten lipomas (10%) revealed both HMGA2-LPP and LPP-HMGA2 fusion transcripts, nine (9%) only HMGA2-LPP, and three (3%) only LPP-HMGA2. DNA sequencing analysis demonstrated that the HMGA2-LPP transcript in 19 lipomas consisted of exons 1-3 of HMGA2 and exons 9-11 of LPP, which was described previously. Out of 13 lipomas with LPP-HMGA2 transcript, 9 were associated with a previously reported LPP-HMGA2 fusion transcript, which fuses exon 8 of LPP to exon 4 of HMGA2, while 4 with a novel type of LPP-HMGA2 fusion transcript, which fuses exon 7 of LPP to exon 4 of HMGA2. CONCLUSION: In addition to the HMGA2-LPP fusion gene, the LPP-HMGA2 fusion gene could have some specific roles for lipomagenesis. The biological implications of the expression and the variation of LPP-HMGA2 fusion transcripts need to be elucidated.

Os odontoideum in achondroplasia: a case report.

The neurological complication in achondroplasia has been focused on the foramen magnum stenosis. We report the combination of os odontoideum in a patient with achondroplasia.

Sp1 family of transcription factors regulates the human alpha2 (XI) collagen gene (COL11A2) in Saos-2 osteoblastic cells.

UNLABELLED: Genes encoding type XI collagen, normally associated with chondrogenesis, are also expressed by osteoblasts. By studying Saos-2 cells, we showed that the transcription factors, Sp1, Sp3, and Sp7 (Osterix), regulate COL11A2 expression through its proximal promoter. The findings indicate both ubiquitous and osteoblast-specific mechanisms of collagen gene regulation. INTRODUCTION: Type XI collagen is essential for skeletal morphogenesis. Collagen XI gene regulation has been studied in chondrocytes but not in osteoblasts. MATERIALS AND METHODS: We cultured Saos-2 cells, a human osteosarcoma-derived line of osteoblasts, and analyzed them for alpha2(XI) protein and COL11A2 regulatory mechanisms. RESULTS AND CONCLUSIONS: Although types I and V were the dominant collagens deposited by Saos-2 cells, they expressed COL11A2 mRNA, and alpha2(XI) chains were present in the extracellular matrix. The COL11A2 promoter region (from -149 to -40) containing three Sp1 binding sites was required for promoter activity in transient transfection assays. All three Sp1 sites were critical for binding by nuclear proteins in electrophoretic mobility shift assays. Further analysis using consensus oligonucleotides and specific antibodies as well as chromatin immunoprecipitation assay implicated Sp1 and Sp3 in binding to this promoter region. Overexpressing Sp1 or Sp3 significantly increased COL11A2 promoter activity and endogenous COL11A2 gene expression, an effect that was suppressed by the Sp1-binding inhibitor mithramycin A. Further experiments showed that Sp1, Sp3, CREB-binding protein (CBP), p300, and histone deacetylase (HDAC) were physically associated and HDAC inhibitors (trichostatin A or NaB) upregulated COL11A2 promoter activity and endogenous gene expression. Another Sp1 family member, Sp7 (Osterix), was expressed in Saos-2 cells, but not in chondrocytes, and was shown by chromatin immunoprecipitation to occupy the COL11A2 promoter. Overexpressing Sp7 increased COL11A2 promoter activity and endogenous gene expression, an effect also blocked by mithramycin A. Using siRNA to knockdown Sp1, Sp3, or Sp7, it was shown that depression of any of them decreased COL11A2 promoter activity and endogenous gene expression. Finally, primary cultures of osteoblasts expressed COL11A2 and Sp7, upregulated COL11A2 promoter activity and endogenous gene expression when Sp1, Sp3, or Sp7 were overexpressed, and downregulated them when Sp1, Sp3, or Sp7 were selectively depressed. The results establish that Sp1 proteins regulate COL11A2 transcription by binding to its proximal promoter and directly interacting with CBP, p300, and HDAC.

Bisphosphonate modulates morphological and mechanical properties in distraction osteogenesis through inhibition of bone resorption.

Despite the general clinical acceptance of distraction osteogenesis and much attention to bone formation in this method, little is recognized about activated bone resorption in the regenerated bone. The purpose of this study was to demonstrate the simultaneously activated bone resorption with activated bone formation and to investigate the role and efficacy of bisphosphonate in distraction osteogenesis. Left tibiae of 54 immature rabbits were lengthened for 3 weeks at a rate of 0.7 mm/day after a 1-week lag. Regenerated bone was quantitatively investigated by radiographic bone density, bone histomorphometry, and three-point bending testing. Animals received either vehicle or nitrogen-containing bisphosphonate (N-BP), YM529/ONO5920 at doses of 0.4 mg/kg/w or 0.004 mg/kg/w for 6 weeks. Regenerated bone of the vehicle group showed a radiologically characteristic zone structure containing the osteopenic zones adjacent to the sclerotic zones. The regenerated bone of the 0.4-mg/kg/w group showed no osteopenic zones during the course and eventually became homogeneously radiodense. The bone volume corresponding to the osteopenic zone of this group was 5.6-fold greater compared with that of the vehicle group. The lengthened bone strength of this group was 3.3-fold greater in ultimate force than that of the vehicle group and equivalent to the contralateral tibia. The 0.004-mg/kg/w group had no substantial differences compared with the vehicle group, despite radiological enhancement of the mineralized front as well as somewhat delayed bone resorption. These results demonstrate that not only bone formation but also bone resorption is highly activated in the regenerated bone, implying high bone turnover. Sufficient N-BP caused a notable modulation in morphological properties of the regenerated bone through inhibition of highly activated bone resorption and eventually increased mechanical properties.

MRI characteristics of parosteal lipomas associated with the HMGA2-LPP fusion gene.

BACKGROUND: The magnetic resonance (MR) characteristics of parosteal lipomas with the HMGA2-LPP fusion transcripts are described. PATIENTS AND METHODS: The expression of HMGA2-LPP fusion transcripts was determined using the reverse transcription-polymerase chain reaction method. RESULTS: MR images of two cases with the fusion transcripts, a 56-year-old man and a 50-year-old woman, revealed heterogeneous high signal intensities on T1- and T2-weighted images, showing heterogeneous curvilinear enhancement on fat-suppressed T1-weighted images after Gd-DTPA injection, which resembled those of well-differentiated liposarcomas. CONCLUSION: Since the HMGA2-LPP fusion transcripts are exclusively detectable in benign mesenchymal tumors, testing HMGA2-LPP expression may be useful for differential diagnosis in cases of radiologically-suspected well-differentiated liposarcomas.

Intrafamilial phenotypic diversity in multiple epiphyseal dysplasia associated with a COL9A2 mutation (EDM2).

We describe a Japanese family with an autosomal dominant multiple epiphyseal dysplasia (MED EDM2) showing significant phenotypic diversity among the five affected members. Genomic analysis for COL9A2 identified an Ex3-1A>G heterozygous mutation, which has been proved to result in skipping of exon 3. The proband was a 9-year-old boy, who presented with ulnar club hands due to severe epiphyseal dysplasia in the distal ulnae. Radiological examination showed multiple epiphyseal dysplasias, predominantly involving the knee and the wrist. The hip appeared almost normal. The malalignment of the wrist was successfully treated with a limb lengthening procedure. The phenotype of the asymptomatic 12-year-old brother was similar to, but milder than, that of the proband. The asymptomatic 39-year-old mother, the 35-year-old uncle, and the 65-year-old grandmother with bilateral painful knees showed radiographically mild and severe osteoarthritis of the knee, respectively, and none of them had wrist deformity.

The alpha 2 type IX collagen gene tryptophan polymorphism is not associated with rheumatoid arthritis in the Japanese population.

The aim of this study was to investigate whether the alpha 2 type IX collagen (COL9A2) polymorphism that introduces tryptophan residue into the collagen triple-helix is a marker of susceptibility to, or severity of, rheumatoid arthritis (RA). The study included 749 Japanese patients with RA. One hundred twenty-four unrelated healthy individuals served as the control subjects. The relationship between the COL9A2 gene polymorphism and clinical manifestations of RA was evaluated. For the number of subjects positive for COL9A2 tryptophan polymorphism, there was no statistically significant difference between RA patients and normal controls. Furthermore, we did not detect any association of COL9A2 tryptophan polymorphism with disease status, least erosive subset, more erosive subset, or mutilating disease. The lack of association of COL9A2 tryptophan polymorphism with RA and the clinical findings in our study implies that the polymorphism may not function as a candidate gene marker for screening RA patients.

[Type IX collagen diseases].

Type IX collagen is a structural protein which consists of the cartilage collagen II/IX/XI heteropolymer. Transgenic mice overexpressing a mutant alpha 1 (IX) collagen chain in cartilage develop a chondrodysplasia-like phenotype associated with early-onset osteoarthritis and spondylosis. Subsequent identifications of the human type IX collagen diseases proved the correctness of the work on mice. Now we know that type IX collagen gene mutations cause multiple epiphyseal dysplasia, an osteochondrodysplasia which frequently features early onset osteoarthritis. Polymorphic variants of the type IX collagen genes are associated with lumbar disc diseases. It is getting more important to understand the biological roles of collagen IX in the musculoskeletal system.

A Human Amnion-Derived Extracellular Matrix-Coated Cell-Free Scaffold for Cartilage Repair: In Vitro and In Vivo Studies.

OBJECTIVE: Extracellular matrix (ECM) derived from human amniotic mesenchymal cells (HAMs) has various biological activities. In this study, we developed a novel HAM-derived ECM-coated polylactic-co-glycolic acid (ECM-PLGA) scaffold, examined its property on mesenchymal cells, and investigated its potential as a cell-free scaffold for cartilage repair. MATERIALS AND METHODS: ECM-PLGA scaffolds were developed by inoculating HAM on a PLGA. After decellularization by irradiation, accumulated ECM was examined. Exogenous cell growth and differentiation of rat mesenchymal stem cells (MSCs) on the ECM-PLGA were analyzed in vitro by cell attachment/proliferation assay and reverse transcription-polymerase chain reaction. The cell-free ECM-PLGA scaffolds were implanted into osteochondral defects in the trochlear groove of rat knees. After 4, 12, or 24 weeks, the animals were sacrificed and the harvested tissues were examined histologically. RESULTS: The ECM-PLGA contained ECM that mimicked natural amniotic stroma that contains type I collagen, fibronectin, hyaluronic acid, and chondroitin sulfates. The ECM-PLGA showed excellent properties of cell attachment and proliferation. MSCs inoculated on the ECM-PLGA scaffold showed accelerated type II collagen mRNA expression after 3 weeks in culture. The ECM-PLGA implanted into an osteochondral defect in rat knees induced gradual tissue regeneration and resulted in hyaline cartilage repair, which was better than that in the empty control group. CONCLUSION: These in vitro and in vivo experiments show that the cell-free scaffold composed of HAM-derived ECM and PLGA provides a favorable growth environment for MSCs and facilitates the cartilage repair process. The ECM-PLGA may become a "ready-made" biomaterial for cartilage repair therapy.

Genomic structure and expression of the mouse ESET gene encoding an ERG-associated histone methyltransferase with a SET domain.

ESET (ERG-associated protein with a SET domain, also called SETDB1) is a novel histone methyltransferase that catalyzes methylation of histone H3-lysine 9 (H3-K9). Here we describe the genomic structure and expression of the mouse ESET gene that gives rise to ESET protein and its alternative splicing product. ESET is a 36-kb single copy gene and full-length ESET transcript consisting of 22 exons. The splicing variant retains only the first 12 exons and thus lacks sequences encoding the methyl CpG-binding domain and the catalytic SET domain. The U2 type conserved GT/AG consensus sequence is present at all of the splicing junctions within the ESET gene. The transcription initiation site of the ESET gene was determined by 5'-RACE experiment and by primer extension. The 5'-flanking sequence of the ESET gene does not contain the consensus TATA box. Instead, this ESET promoter region has features such as SP1-binding sites that are typical of housekeeping genes. The ESET promoter was functionally active when tested in transfection and luciferase assay. Full-length ESET transcript appears to be ubiquitously expressed. While the SET domain-deficient splicing variant is present in immortalized cell lines, it is undetectable by RT-PCR in the majority of normal mouse tissues.

Differential expression of VEGF isoforms and VEGF receptors in cartilaginous tumors.

Vascular endothelial growth factor (VEGF), which is known to have at least five isoforms and four receptors, is integral to tumor-induced neovascularization. In order to determine whether VEGF signaling is modulated in the development of cartilaginous tumors, we analyzed osteochondrogenic tumors for the expression of VEGF isoforms and VEGF receptors using the reverse transcription-polymerase chain reaction method. Although mRNA for VEGF121, VEGF165, VEGFR-1 and NRP-2 was widely expressed, mRNA expression for VEGF189, VEGFR-2 and NRP-1 was linked to a malignant phenotype such as local invasion. These results indicated that VEGF signaling was fine-tuned by the differential expression of the VEGF isoforms and VEGF receptors.

Matrix deposition of tryptophan-containing allelic variants of type IX collagen in developing human cartilage.

Genetic polymorphisms that encode a tryptophan (Trp) residue in the triple-helical domain of the alpha2 (Trp2) or alpha3 chain (Trp3) of human type IX collagen have been linked to risk of degenerative intervertebral disc disease. To determine whether these two allelic variants express protein that may affect the extracellular matrix of cartilage in vivo, we examined the properties of resident type IX collagen in an anonymous collection of embryonic and fetal human cartilage samples screened for Trp genotypes. No difference was found in the yield and electrophoretic properties of pepsin-solubilized type IX collagen between Trp2, Trp3 and non-Trp cartilage samples. On Western blot analysis, a polyclonal antiserum raised against a synthetic peptide matching the immediate Trp-containing sequence of the Trp3 allele reacted specifically with the alpha3(IX) chain prepared from Trp3 cartilage samples. Two-dimensional peptide mapping of type IX collagen in CNBr-digests of whole tissue gave indistinguishable fingerprints for Trp2, Trp3 and control tissues, including the yield of cross-linked peptides. Analysis of one cartilage sample that was homozygous for the Trp2 allele also gave a normal yield of collagen IX, including its alpha2 chain and a normal profile of cross-linked peptides. Together, the findings indicate that both Trp2 and Trp3 allelic products are incorporated into the cross-linked fibrillar network of developing human cartilage apparently normally. Any pathological consequences are likely, therefore, to be long-term and indirect rather than from overt misassembly of matrix.